title: A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification.
abstract: Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×10(4)M(-1)s(-1). It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10(-4)M(-1)s(-1). The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of a thermophilic arsenate reductase.
authors.affiliation: Department of Biology, University of Naples Federico II, Edificio 7, via Cinthia, 80126 Naples, Italy.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Del Giudice
authors.forename: Danila
authors.initials: D
authors.lastname: Limauro
authors.forename: Emilia
authors.initials: E
authors.lastname: Pedone
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bartolucci
authors.forename: Gabriella
authors.initials: G
authors.lastname: Fiorentino
mesh_terms: D000595:Amino Acid Sequence; D053502:Arsenate Reductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001149:Arsenates / Q000737:chemistry* / Q000378:metabolism; D018053:Arsenites / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001149:Arsenates; D018053:Arsenites; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D053502:Arsenate Reductases; C015001:arsenite; C025657:arsenic acid
keywords: (Thermus medium); (arsenate respiratory reduction); (arsenic resistance system); (arsenite oxidation system); (gene ID: 2776273); (low molecular weight protein tyrosine phosphatases); (minimal inhibitory concentration); (p-nitrophenyl phosphate); (thioredoxin reductase); (thioredoxin); Arsenate reductase; Arsenate resistance; Hyperthermophile; LMW-PTPase; MIC; Protein thermal stability; TM; Tr; Trx; TtarsC; aox; arr; ars; pNPP